Investigation of combination of conventional culture and micro culture method for Helicobacter pylori isolation and antigen preparation from culture for using in serologic diagnosis

H. pylori is recognized as an important pathogen with high prevalency which can cause various diseases such as gastric cancer, peptic ulcers and gastric lymphoma. At the same time H. pylori infection is a serious public health problem in Turkey. Turkey has a 85% prevalence of infection among patients with dyspepsia. H. pylori biopsy specimen isolation, culture obtaining and producing with a high amounts are important due to the development of new drugs, vaccines also the study of the diagnostic methods and mechanisms of resistance. For the isolation of H. pylori generally dilution and classic culture method are being used. However, the use of existing methods is not sufficient to achieve long-term and culture and the adaptation of the bacteria to the culture medium. It?s known that the success rate of conventional culture method is about 55% for H. pylori diagnosis and isolation. One of the main challenge is microaerophilic environmental conditions are needed for production of H. pylori. In the literature the it?s demonstrated that the micro culture method is utilizable in order to provide microaerophilic environment. However, there is no study about micro-culture method for the isolation of H. pylori. On the other hand, ELISA which is one of the advantageous world-wide method in terms of cost sensitivity and specificity of studies reporting conflicting results. These contradictory results preoccupies that the antigenic material used in commercial kits should be different from region-specific strains antigenic structures. In addition, Despite our country has high prevalence of H. pylori than other infections, diagnostic kits are being imported from abroad. So far, In our country, there is no study about preparation of ELISA diagnostic kits based on local H. pylori strain?s antigens. In this sense, the preparation of domestic ELISA kits and their efficiency comparisons with commercial kits is very important to diagnose with high accuracy against this public health problem in our country. According to all information, the aim of this thesis, for the first time, H. pylori isolation is made by Micro Culture Method, which used in the isolation of Leishmania parasites previously, to increase the sensitivity of classical culture. To obtain a continuous culture of H. pylori and biomass. H. pylori isolation, are used to make the diagnosis of classical culture, and culture in order to increase the sensitivity of the method, previously used by using micro-Culture Method, to increase the sensitivity of the method, isolated from H. to obtain a continuous culture of H. pylori, and the resulting culture pylori antigen for use in serological diagnosis based on the ELISA test has been developed to compare the commercial kits. Patient samples which are used in the study were provided by Department of Clinical Microbiology of Istanbul University Faculty of Medicine. Micro culture method was applied in combination with classical culture isolation. After incubation for 48 hours under conventional culture was started. Than, isolation rates compared with conventional culture method. The obtained colonies were characterized by PCR and Gram staining. Continuous culture of isolated bacteria were obtained and Flow cytometric viability was studied. The biomass of culture was obtained and bacterial antigens were obtained. ELISA test desinged with obtained antigen and the sensitivity of that ELISA test was compared with commercial ELISA kit. For the firs time in this study, a new protocol about isolation H. pylori from biopsy samples by Micro Culture Method has been developed. The combination of Micro Culture Method and classical culture more sensitive than classical culture by 30%. 1,7565 mg soluble antigen was obtained from biomass. Finally, designed ELISA test?s sensitivity was 82,4%, commercial ELISA kit?s sensitivity was 70,5%. Key words: Helicobacter pylori, Isolation, Conventional Culture, Micro Culture Method, ELISA, Diagnostic kit